Eric Kmiec, director of the Gene Editing Institute, led the team that used CRISPR on DNA in a lab dish. (Christiana Care Health System)

Researchers have been developing CRISPR gene editing as a potential therapeutic tool for years, but its most immediate use may be a less obvious one: cancer diagnostics. In what they say is a first for the gene-editing technology, researchers from Christiana Care Health System used CRISPR to edit DNA outside the cell, laying the groundwork for a diagnostic test that can replicate DNA mutations in a patient's tumor and help physicians identify the most appropriate treatment.

The team, led by Eric Kmiec, the director of Christiana Care's Gene Editing Institute, edited genes inside plasmids, a type of DNA that can be removed from a cell and manipulated in a lab dish. They started out using Cas9, the backbone of many CRISPR systems, but it proved ineffective at editing DNA outside the cell. When they switched to a different enzyme, Cpf1 (also called Cas12a) "it changed everything," Kmiec said in an interview with FierceBiotechResearch.

When Cas9 cuts DNA, it does so like a sharp pair of scissors, leaving "blunt ends." Kmiec said. Because the ends are so smooth, it requires precise positioning to insert replacement DNA. In contrast, Cas12a leaves frayed, or "sticky," ends. There are single-stranded "DNA overhangs" that act like velcro, making it much easier to add DNA fragments to the sequence. The study appears in The CRISPR Journal, which published its inaugural issue in February.

When cancer patients are diagnosed, their tumors are often sequenced to identify genetic mutations that are key drivers of their cancer. The CRISPR-Cas12a system can be used to replicate DNA mutations based on this sequencing information, Kmiec said. Recreating them on synthetic pieces of DNA gives researchers a look at what pathways might be affected by mutations, he added.

While a PCR-based method called site-directed mutagenesis already exists to recreate mutations in vitro, using CRISPR could significantly accelerate the process, Kmiec said. The CRISPR-Cas12a system can also make multiple changes to DNA samples in one pass, while PCR systems require multiple rounds to replicate multiple mutations.

Kmiec's team is now working on a "CRISPR-on-a-chip" for a commercial partner that has licensed the technology for cancer diagnostics. Though diagnostics is the most immediate use for the tech, the Gene Editing Institute continues to work on therapeutic CRISPR with programs in lung cancer and melanoma. Current CRISPR systems can only make edits on a single gene; down the line, Kmiec's work could lead to a CRISPR therapy that treats diseases like Alzheimer's that involve mutations in several genes.

CRISPR is already being investigated for diagnostic purposes. Scientists at the Broad Institute are developing a Cas13a-based system to test for Zika virus in blood, urine or saliva. The tool, dubbed SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), targets RNA rather than DNA, and doesn’t stop at cutting its intended target. Instead, it also cuts RNA that happens to be nearby, which the scientists called “collateral cleavage.”

Kmiec says another advantage of using CRISPR outside the cell is that it could help demystify how, exactly, the technology makes its edits.

“When you’re working with CRISPR inside a cell, you’re kind of working in a black box where you can’t really observe the gears of the machinery that are doing these amazing things,” Kmiec said in a statement. “You can see the results, the edits to the genes, but not necessarily how you got there, which is important for ensuring that CRISPR can be safely used to treat patients.”

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